Chemistry Department, University of Nebraska, Lincoln, NE 68588-0304, USA.
Anal Bioanal Chem. 2012 Apr;403(2):563-71. doi: 10.1007/s00216-012-5816-y. Epub 2012 Feb 22.
Columns containing immobilized low-density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nK(a)) of 1.9 (±0.1) × 10(5) M(-1) for R-propranolol and 2.7 (±0.2) × 10(5) M(-1) for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (K(a)) of 5.2 (±2.3) × 10(5) M(-1) at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents.
采用高效亲和色谱（HPAC），为分析药物与这种试剂的相互作用，制备了固定化低密度脂蛋白（LDL）的柱子。R/S-普萘洛尔被用作本研究的模型药物。在 pH 7.4、0.067 M 磷酸钾缓冲液存在的情况下，LDL 柱子在连续使用 60 小时后，对普萘洛尔仍具有可重复的结合作用。通过前沿分析进行的此类柱子实验表明，R-普萘洛尔和 LDL 之间发生了两种类型的相互作用，而 S-普萘洛尔和 LDL 之间仅观察到一种类型的相互作用。第一种相互作用（两种对映体都有）涉及非饱和结合；这种相互作用的总亲和力（nK(a)）对于 R-普萘洛尔为 1.9（±0.1）×10(5) M(-1)，对于 S-普萘洛尔为 2.7（±0.2）×10(5) M(-1)，温度为 37°C。第二种相互作用仅在 R-普萘洛尔中观察到，涉及饱和结合，其在 37°C 时的缔合平衡常数（K(a)）为 5.2（±2.3）×10(5) M(-1)。在 20°C 和 27°C 时，两种对映体的结合行为也存在类似的差异。这是 LDL 或其他脂蛋白对药物进行立体选择性结合的首例已知实例。这项工作还说明了 HPAC 可作为一种工具，用于表征涉及 LDL 和相关结合剂的混合模式相互作用。
